1. Field of the Invention
Embodiments of the invention are most generally related to the field of fluorescence emission and collection and/or imaging and/or non-linear harmonic emission collection and/or imaging; collection and/or imaging optical apparatus/systems such as, but not limited to microscopes, endoscopes, and laparoscopes, operational component apparatus thereof, associated methods, and applications. More particularly, embodiments of the invention are directed to optical scanning component apparatus and imaging system components, and associated methods and applications of use.
2. Description of the Related Art
The multiphoton microscope was co-invented almost two decades ago by Dr. Watt Webb, a co-inventor of the present invention. Multiphoton microscopy (MPM), as is now well known, is a special kind of laser scanning microscopy that provided significant advantages over standard confocal microscopy. In confocal microscopy, one photon of high energy light (at, e.g., 488 nm) is used to excite a molecule to produce one photon of fluorescence. The light excites molecules in a relatively large region around the focal point. The use of high energy light could easily damage living tissue in the entire region of exposure. Furthermore, imaging depth was limited to about 50 microns (μ) (about five cell layers).
In MPM, multiple lower energy photons (at, e.g., 780-800 nm, 960 nm, other suitable excitation wavelengths) impinge on a fluorescent target molecule simultaneously, producing one photon of fluorescence from the focal volume of the excitation field. Advantageously, MPM is safer and more efficacious than confocal microscopy for human use because of, e.g., limited site photo-toxicity and photo-damage to living tissue, imaging depths up to 500 to 1000 μm, and lower out-of-focus fluorescence background. Intrinsic fluorescence of certain tissue structures generated by the excitation field reduces or eliminates the need for dye (fluorophore) injection. There are other reasons known in the art. As a result, MPM provides the capability to acquire high contrast, high resolution images, without the need to use pinhole apertures or other spatial filtering elements, with reduced tissue photo-bleaching and photo-destruction that occur from repeated excitation.
The laser light used (typically femtosecond (fs) pulses) to generate multiphoton-induced excitation also supports the non-linear optical phenomenon known as harmonic generation. Second harmonic generation (SHG) (and higher-order harmonic generation) under multiphoton excitation can cause collagen and certain tissue structures such as microtubule bundles, nerves and cartilage, for example, to emit intrinsic SHG radiation.
The present inventors have recognized that various advantages and benefits could be realized by incorporating the principles of laser scanning microscopy into medical instruments such as surgical microscopes, and endoscope- and laparoscope-type apparatus for in-situ and in-vivo fluorescence and/or higher-order harmonic emission imaging and/or fluorescence and/or higher-order harmonic emission collection. In addition, advances made in the course of developing such medical instruments are useful in instruments designed for research or for industrial microscopy. In the medical realm, disease diagnosis has for a long time been, and continues to be, carried out by various biopsy procedures. A biopsy requires the physical removal of a (deep) tissue sample from a patient, sample analysis by a pathologist, and reporting, which may take from a few hours to several days or more. The ability to perform real time, in-situ and in-vivo endoscopy in combination with the diagnostic capabilities of fluorescence and harmonic-scattering-based imaging could significantly reduce the pain, time, and cost associated with conventional biopsy procedures and assist in disease diagnosis and the extent of tissue damage due to disease states. High resolution laser scanning endoscopy, laparoscopy, or surgical microscopy for sub-tissue, nerve, and cartilage examination offers advantages over the capabilities of current instruments. The ability to see nerves and collagen clearly would be especially valuable, for example, in nerve-sparring prostate surgery, bladder cancer treatment, maxillofacial and oral surgery, and other surgical and diagnostic applications for animals and humans.
Miniaturized instruments capable of confocal, optical coherence tomography (OCT), two-photon fluorescence (TPF), and second harmonic generation (SHG) imaging have been reported. The typical constituents of these devices include a miniaturized scanning mechanism and a lens assembly that is encapsulated in a protective housing with dimensions suitable for use in small spaces (e.g., in minimally invasive medical procedures) have been described; for instance, a probe outer diameter on the order of a few millimeters with a rigid length of several centimeters. Within these laser scanning microscopes (similarly, laparoscopes/boroscopes and other microscopes), various distal miniaturized scanners have been demonstrated, including resonant-based (e.g., Lissajous or spiral scan pattern) and non-resonant-based, cantilever fiber scanners, as well as microelectromechanical systems (MEMS) scanning mirrors. Of these scanners, the resonant-based spiral scanners are the most successful in terms of their miniaturized dimensions (e.g., OD≈1 mm) and fast image acquisition speeds (e.g., 8 frames/s with 512×512 pixels per frame, ≈200 μm diameter FOVxy); however, these resonant devices are fundamentally limited by non-uniform spatial coverage and sampling time in comparison to current miniaturized raster scanners. Current miniaturized raster scanners are, however, limited in terms of their physical dimensions and/or scan speeds. Le Harzic et al. has previously demonstrated a piezo-driven X-Y scanner (length=34 mm, width=1.9 mm) capable of a uniformly sampled FOVxy up to 420 μm by 420 μm, but this device is limited by its frame rate (i.e., 0.1 frames/s with 512×512 pixels per frame) (Le Harzic R, Weinigel M, Riemann I, Konig K, Messerschmidt B (2008) Nonlinear optical endoscope based on a compact two axes piezo scanner and a miniature objective lens, Opt Express 16:20588-20596). Slow image acquisition speeds are not ideal due to the motion artifacts typically faced in real-time in vivo clinical imaging environments. Additionally, although 2-D MEMS scanning minors with miniature dimensions (e.g., 750 μm×750 μm mirror size) have recently demonstrated fast line acquisition rates on the order of 1-3 kHz, the overall miniaturization of these MEMS scanners (i.e., their probe ODs) is limited by the die size of the actuator, which is typically 3 mm×3 mm.
Furthermore, in any scanning system there is a fundamental relationship between resolution (independent pixels) and the scanner range. It is often the case that the relationship between the output beam size and the number of resolvable points is fundamental and cannot be altered by external optics.
Efforts to date to improve endoscopic and laparoscopic imaging procedures and apparatus have focused on the laser scanning fluorescence excitation processes with little attention directed to improved systems and methods for acquiring, identifying, and analyzing the fluorescence, or to improved systems and methods to reduce the severity and invasiveness of existing procedures.
In-vivo laser scanning microscopy, including multiphoton microscopy (MPM), has become a valuable tool for the study of deep structures in intact animals. MPM through gradient index (GRIN) lens systems has been shown to be useful in relaying the excitation light and autofluorescence/second harmonic generation (SHG) emission to and from an external microscope deep into soft tissue. These studies have, however, been limited to short (<3 cm) GRIN systems for small animal use. With MPM showing great promise in the diagnosis of human diseases, many applications (e.g., lung, prostate, bladder, other, examination/diagnosis,) will require significantly longer GRIN systems.
In view of the problematic challenges and shortcomings associated with fluorescence emission (endoscopy, laparoscopy, and microscopy) imaging/collection apparatus and methods, the inventors have recognized the unfulfilled need for apparatus and methods that can address and solve these challenges and shortcomings, and others, in a practical, cost effective, and efficacious manner.
Embodiments of the invention are directed to apparatus and methods that address the aforementioned problems associated with current technology in this field.